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Chroococcidiopsis Geitler (Geitler 1933) is a genus of cyanobacteria containing desiccation and radiation resistant strains. Members of the genus live in habitats ranging from hot and cold deserts to fresh and saltwater environments. Morphology and cell division pattern have historically been used to define the genus. To better understand the evolution and ability of the Chroococcidiopsis genus to survive in diverse environments we investigated how salt tolerance varies among 15 strains previously isolated from different locations, and if salt tolerant strains are monophyletic to those isolated from freshwater and land environments. Four markers were sequenced from these 15 strains, the 16S rRNA, rbcL, desC1, and gltX genes. Phylogenetic trees were generated which identified a distinct clade of salt-tolerant strains. This study demonstrates that the genus is polyphyletic based on saltwater and freshwater phenotypes. To understand the resistance to salt in more details, the strains were grown on a range of sea salt concentrations which demonstrated that the freshwater strains were salt-intolerant whilst the saltwater strains required salt for growth. This study shows an increased resolution of the phylogeny of Chroococcidiopsis and provides further evidence that the genus is polyphyletic and should be reclassified to improve clarity in the literature.